Prediction, screening and characterisation of novel bioactive, tetra-peptide matrikines 2
Prediction, screening and characterisation of novel bioactive, tetra-peptide matrikines 2
The effects of the peptide blend was assessed by RNA-Seq analysis. Primary human dermal fibroblast cells from adult Caucasian females aged between 23-27 (taken from buttock (n=3)) were seeded within appropriate cell densities into tissue culture plates in assay medium – Fibroblast Basal medium 2 (phenol red-free; PromoCell, C-23225) containing 2% FBS, 0.001% basic human fibroblast growth factor (hbFGF) and 0.005% insulin (Fibroblast growth medium 2 supplement pack; PromoCell, C39320)). Primary human dermal fibroblast cells from adult Caucasian females aged between 23-27 (taken from buttock (n=3)) were seeded within appropriate cell densities into tissue culture plates in assay medium – Fibroblast Basal medium 2 (phenol red-free; PromoCell, C-23225) containing 2% FBS, 0.001% basic human fibroblast growth factor (hbFGF) and 0.005% insulin (Fibroblast growth medium 2 supplement pack; PromoCell, C39320)).  After two days of culture in a humidified environment at 37°C with 5% CO2, once cells had reached confluency, cells were either left untreated as a negative control, or treated with a combination of P1 and P7 at 8 parts per million (ppm) each, or 1 μM ATRA (Sigma-Aldrich, R2625). All treatments were performed in triplicate. RNA was extracted 12 hours following the treatment using the RNeasy Mini Kits (74104, Qiagen) according to the manufacturer's instructions. Briefly, lysis buffer was added to the samples, followed by precipitation of nucleic acids and desalting by addition of 70% ethanol. The resulting mixture was added to the RNeasy Mini Spin Columns and centrifuged to bind total RNA to the membrane. Samples were then washed, first with buffer RW1 to remove biomolecules such as carbohydrates, proteins and fatty acids which may have bound non-specifically to the membrane. The membrane was then washed twice with buffer RPE to remove salts from previous steps of the method. Finally, using RNAse-free water, total RNA was eluted from the spin columns. All extraction steps were performed on ice, with pre-chilled solutions. Following RNA extraction, RNA sample concentration was measured using a NanoDropTM Spectrophotometer (ThermoFisher Scientific). All samples were stored at -80˚C, and repetitive freeze-thaw cycles were avoided by aliquoting RNA samples. RNA samples were analysed as described below. Stranded mRNA assay (Illumina)